EFFECT OF FLUORIDE AND GONADAL STEROID DEFICIENCY ON ENAMEL AND DENTIN MINERALIZATION OF FEMALE RATS

1Undergraduate Student, Araçatuba Dental School, São Paulo State University, Araçatuba, São Paulo, Brazil. 2Assistant Professor of the Discipline of Physiology of the Department of Basic Sciences, Araçatuba Dental School, São Paulo State University, Araçatuba, São Paulo, Brazil. 3Associate Professor of the Discipline of Pediatric Dentistry of the Department of Pediatrics and Social Dentistry of Araçatuba Dental School, São Paulo State University, Araçatuba, São Paulo, Brazil. 4Assistant Professor of the Discipline of Biochemistry of the Department of Basic Sciences, Araçatuba Dental School, São Paulo State University, Araçatuba, São Paulo, Brazil.


INTRODUCTION
Hormones and growth factors can influence the metabolism of ameloblasts and odontoblasts 1,21,23 .However, the role played by sexual hormones in the process of tooth development remains scarcely investigated.During the 1970s, Spiegel, et al. 25  (1971) observed that puberty disorders led to mild alterations in tooth development.Other studies have demonstrated that periods of pregnancy and lactation 18 , as well as ovariectomy (OVX) 8 , lead to alterations in the rate of dentin apposition in female rats.Moreover, Jukic, et al. 13  (2003)  detected the presence of estrogen receptors in human dental pulp.Also, Hietala, et al. 11 (1998) visualized, by immunohistochemical staining, estrogen-receptor-related antigens in the predentinal-odontoblast region of human teeth scrapings.
Ovariectomy in female rats is an experimental model that simulates the post-menopause period in women.It causes osteoporosis with reduction in bone mass and the alterations in its microstructure characterize the osteoporosis and increase the susceptibility to fractures 14 .
Fluoride participates in the process of enamel mineralization in vivo and influences the structure and properties of the mineral formed.During amelogenesis, the presence of free fluoride ions accelerates the formation of apatite crystals 27 .Fluoride replaces the hydroxyl group of apatite and leads to a reduction in the volume of the crystal and increase in the structure stability 2 .However, high concentrations of fluoride may cause disturbances in enamel maturation, leading to subsurface hypomineralization 28 .
The aim of this study was to verify whether the gonadal steroid deficiency and the chronic administration of fluoride can influence the process of mineralization of the enamel and dentin in maxillary incisors of female rats.

MATERIAL AND METHODS
Forty-four female rats (Wistar/160-180g), kept at a lightcontrolled (12 hours of dark -12 hours of light) and temperaturecontrolled (22 ± 2°C) environment, were submitted to ovariectomy (OVX group) or sham ovariectomy (sham group) after anesthesia with chloral hydrate (4 mg/Kg, IP).The animals received commercially available balanced pellets (Guabi nutrilabor, Mogiana Alimentos S.A.) and water ad libitum.The study design was approved by the Ethics Committee for Animal Experimentation of the Araçatuba Dental School, UNESP.After 15 days, half of each group was given distilled water (sham and OVX groups) or distilled water with 10 ppm fluoride as NaF (sham/NaF and OVX/NaF groups).The animals were killed with excess ethyl ether inhalation and the maxillary incisors were extracted ninety days after onset of treatment with water or fluoride solution.The incisors were embedded in acrylic resin and the specimens were ground, polished and submitted to microhardness analysis in longitudinal sections, employing the microhardness meter Shimadzu HMV-2.000™, with a Knoop indenter under a static load of 50 g for 10 seconds 23 .Four indents were performed on the middle portion of enamel thickness: the first was performed at 1 mm from the incisal end; the second at 2 mm from the first indent, towards the root; the third at 1 mm from the root apex; the fourth at 2 mm from the third indent towards the crown.Regarding the dentin, the indents were performed in the same direction as for enamel, at 150 µm from the dentin-enamel junction.
After confirmation of the homogeneity of the data, the results were submitted to analysis of variance and Tukey's test on the GMC software 5 , at a significance level of 5%.

RESULTS
The data from the incisal and middle regions of the crown, as well as on the apical and cervical regions of the root were gathered in a single block to allow a better analysis of the microhardness of enamel and dentin.The means and standard errors (SEM) of enamel and dentin microhardness are graphically displayed (Figures 1 and 2, respectively).
Analysis of enamel microhardness of the maxillary incisors (Figure 1) showed that OVX animals had a significant reduction in the mineralization of crown (7.9% and 8.1%) and root (20.4% and 25.0%) in both groups treated with fluoride or distilled water, respectively.However, the sham animals did not display any significant alteration in enamel microhardness after chronic administration of fluoride.

EFFECT OF FLUORIDE AND GONADAL STEROID DEFICIENCY ON ENAMEL AND DENTIN MINERALIZATION OF FEMALE RATS
The castrated animals exhibited higher (14.2%) dentin microhardness at the crown (Figure 2), yet the 90-day treatment with fluoridated distilled water precluded this increase.Moreover, the increase in microhardness of root dentin of rats submitted to sham ovariectomy and treated with fluoride was not observed in the OVX animals who received the same treatment.

DISCUSSION
There is a lack of investigations on the effects of estrogen deficiency secondary to menopause on the metabolism of odontoblasts or ameloblasts in humans since menopause occurs when teeth are already formed.Therefore, the studies found in the literature are related to investigations conducted in female rats submitted to ovariectomy 29 .Rats' incisors present continuous growth and constitute an interesting model for investigation of the influence of several factors on the different stages of odontogenesis.
In the present study, the reduction in microhardness of the incisor enamel observed in ovariectomized rats (Figure 1) indicates that estrogens may directly or indirectly influence the process of enamel mineralization of these teeth.Estrogen deficiency leads to disturbances in calcium and phosphate homeostasis, which may affect dental tissues 10,16,17 .Microhardness is a physical property that depends on the calcium and protein content in enamel and dentin.According to Lozupone and Favia 17 (1994), the enamel microhardness in incisors of rats submitted to a calcium-deficient diet is reduced, even though a normal amount of organic matrix has been formed.The estrogen deficiency secondary to ovariectomy leads to a lower intestinal absorption of calcium 14 and to a decrease in the serum levels of calcium ions 12 .This may explain the deficient enamel mineralization, leading to a reduction in microhardness.
On the other hand, the administration of fluoride at a concentration of 10 ppm in the drinking water for 90 days did not preclude the reduction in enamel microhardness induced by ovariectomy.In fact, it displayed a tendency toward further reduction in enamel microhardness, yet without statistical significance.It is known that fluoride incorporated into enamel crystals affects the subsequent mineralization processes because it reduces the mineral solubility and modulates the ionic composition in the fluid around the mineral 3 .In mature enamel, the matrix is replaced by enamel crystals as proteolytic enzymes (metalloproteinases and serine proteinases) degrade it 20 .According to Bawden, et al. 4 (1995), the enamel fluorosis secondary to the intake of high doses of fluoride may be yielded by a disturbance in the removal of amelogenins during the maturation stage, induced by inhibition of proteases by fluoride.DenBesten, et al. 7 (2002) observed a reduction in the activity of proteases in the enamel matrix in incisors of rats treated with 50 and 100 ppm fluoride in drinking water.On the other hand, Gerlach, et al. 9 (2000) observed that fluoride (625 µM to 10 µM) inhibited neither the proteinases activity of the crude enamel matrix extract nor the activity of individual enamel enzymes separated by SDS-PAGE.In fact, the amounts of fluoride required locally to affect an inhibition of amelogenin digestion are unknown.The 10 ppm concentration employed in the present study may not have yielded inhibition of the activity of these enzymes enough to lead to a decrease in mineralization.
Sexual steroids are fundamental for bone maintenance throughout life and the adverse effects of the deficiency of such hormones are noticeable in men and women and in young and old persons.The post-menopause osteoporosis induced by the reduction in the serum concentration of estrogen is characterized by reduction of the bone mass, leading to an increase in fragility and risk of fracture 6 .Laboratory studies have demonstrated that estrogen deficiency caused by ovariectomy in female rats leads to the imbalance between bone formation and resorption causing osteoporosis 29 .Since osteoblasts and odontoblasts have a common mesenchymal origin and both secrete extracellular matrix that will undergo mineralization, the function of both cells is likely to be regulated by the same mechanisms.Moreover, some authors suggest activation of the function of odontoblasts 10 and osteoblasts 29 after castration of female rats.
Although enamel in adults is considered a relatively stable tissue, alterations in dentin may occur as secondary dentin formation or dentinal tubules obliteration due to calcium phosphate crystals deposition.The higher dentin microhardness of the maxillary incisor of OVX female rats (Figure 2) perhaps could have been explained by the deposition of inorganic material.Therefore, it may be suggested that postmenopause deficiency of estrogen in women may affect sclerotic dentin, which is observed in the elderly.
The deficiency of estrogen during menopause causes a reduction in the intestinal absorption of calcium, which in turn leads to an increase in the concentration of parathormone (PTH) 15,26 .Yonaga 30 (1978) studied the effect of this hormone on the mineralization and formation of dentin matrix in incisors of immature rats and observed that these processes were accelerated and did not necessarily occur concurrently when the PTH hypersecretion was caused by a calcium-deficient diet.This may explain the increase in the microhardness of the crown of the incisor of the castrated female rat (Figure 2).Hietala and Larmas 10 (1992) also observed an increase in dentin formation in female rats following ovariectomy.As already mentioned, Hietala, et al. 11 (1998) visualized estrogen-receptorrelated antigens in the predentinal-odontoblast region, which could influence that pattern.
Fluoride has been used daily for osteoporosis treatment in women (50 to 75 mg/day). 22That dosage is similar to that ingested by rats after drinking water with 10 mg F /L (the concentration used in this study).According to Shinoda 24 (1975), 10 ppm F in drinking water did not alter enamel and dentin microhardness of rats' incisors.In the present study, the increase in microhardness of dentin of group OVX was not observed after treatment with fluoride (group OVX/NaF, Figure 2).Moseley, et al. 19 (2003) observed inhibition of collagen synthesis by odontoblasts after fluoride exposure, which would lead to an alteration of the extracelular matrix and of the dentin mineralization.This may explain the results of the present study.
Standardization of the area for hardness measures in root is difficult as rats' incisors present continuous growth.This could have led to measures in regions closer to the crown.This may explain the increase in root microhardness of the incisor of female rats submitted to sham/NaF (Figure 2), when compared to sham group.

CONCLUSION
The results indicate that the sexual steroids influence the process of mineralization of the enamel and dentin in incisors of female rats.Also, it was demonstrated that enamel and dentin exhibit different responses to fluoride administration.However, further investigations are required to explain the mechanisms involved in this process.